Loss of Redox Factor 1 Decreases NF-kappaB Activity and Increases Susceptibility of Endothelial Cells to Apoptosis

OBJECTIVE: The aim of this project was to test the hypothesis that redox factor 1 (Ref-1) was a critical upstream determinant of NF-kappaB-dependent survival signaling pathways in the vessel wall. METHODS AND RESULTS: Aortas from hemizygous transgenic mice harboring a single allele of Ref-1 exhibited a significant loss in NF-kappaB DNA binding activity. The NF-kappaB-dependent survival gene A20 was significantly downregulated in aortas of hemizygous Ref-1 mice, whereas IAP-2 was unchanged. Overexpression of A20 rescued cells from tumor necrosis factor (TNF)-induced apoptosis, suggesting that the loss of A20 in Ref-1 hemizygotes may be a rate-determining step in endothelial cell fate. Deletion of the previously defined redox-sensitive or the AP endonuclease domains of Ref-1 significantly decreased NF-kappaB transcriptional activation and endothelial cell survival. Furthermore, TNF-induced apoptosis was significantly potentiated in endothelial cells after delivery of Morpholino antisense oligodeoxynucleotides targeted to Ref-1. Loss of the redox-sensitive domain blocked the ability of Ref-1 to reduce p50; however, loss of the endonuclease domain did not effect p50 reduction, suggesting alternative mechanisms of action of Ref-1 on NF-kappaB activity. CONCLUSIONS: These findings establish a role for Ref-1 as an upstream determinant of NF-kappaB and A20-dependent signaling and endothelial survival in the vessel wall.